Monocyte activation by HbS is mediated by TLR4. (A) Surface expression of TLR4 on human monocytes from patients with SCD (n = 19) and controls (n = 10), measured by flow cytometry. (B) mRNA expression (relative to β-actin and compared with control) of TLR4 by human monocytes from healthy blood donors (n = 5), in culture with HbS (20 μM) or HbA (20 μM), after 4 hours (n = 5 per group). (C-E) Levels of TNF-α (C), IL-1β (D), and IL-6 (E) in the cell culture supernatant of human monocytes from healthy blood donors (n = 4), in culture with HbS (20 μM), HbS (20 μM) with the TLR4 inhibitor TAK-242 (1 μM) added to culture medium 1 hour before HbS, or control, after 18 hours (n = 4 per group). (F-G) Levels of TNF-α (F) and IL-6 (G) in the cell culture supernatant of THP1-Dual and THP1-Dual KO-TLR4 cells in culture with HbA (20 μM), HbS (20 μM), LPS (0.1 μg/mL), or control, after 18 hours (n = 3 per group). (H-I) The NF-κB pathway activation level (H) and type I interferon pathway activation level (I) relative to control, of THP1-Dual and THP1-Dual KO-TLR4 cells in culture with HbA (20 μM), HbS (20 μM), LPS (0.1 μg/mL), or control, after 18 hours (n = 6 per group). ∗∗∗P < .001; ∗P < .05 by Mann-Whitney test (A) or one-way ANOVA (B-I). MFI, mean fluorescence intensity.